Tetranuclear Schiff base copper(II) complexes: Syntheses, crystal structure, DNA/protein binding and catecholase-like activity

Article abstract of DOI:10.1016/j.poly.2019.01.057

Two tetranuclear Schiff base copper(II) complexes, namely [Cu₄(L)₂(LH)₂(H₂O)₂](ClO₄)₂·3H₂O (1) and [Cu₄(L)₂(LH)₂(H₂O)₂](ClO₄)·(tp)_0.5·3H₂O (2) (where H₂L = 2-[(2-hydroxy-ethylimino)-methyl]-6-methoxy-phenol, tp = terephthalate), were synthesized and characterized by X-ray single crystal diffraction. Both complexes 1 and 2 are comprised of structurally similar tetranuclear cationic [Cu₄(L)₂(LH)₂(H₂O)₂]²⁺ species with a double open cubane core, in which two metal centers possess a square pyramidal environment and the other two exhibit a slightly distorted octahedral coordination geometry. The interaction of complexes 1 and 2 with calf thymus DNA (CT-DNA) was investigated using electronic absorption and fluorescence studies, and the results showed that the complexes interact with CT-DNA with the related intrinsic binding constants (K_ib) of 1.8 × 10⁶ and 1.1 × 10⁷ L mol^?1 for 1 and 2, respectively. Their interactions with bovine serum albumin (BSA) and human serum albumin (HSA) were also investigated and spectroscopic techniques showed that both complexes interact with these proteins through a ground state association process. Using 3,5-di-tertbutylcatechol (3,5-DTBC) as a model substrate, both complexes show catecholase-like activity, being able to oxidase 3,5-DTBC to 3,5-di-tert-butylquinone (3,5-DTBQ) in the presence of oxygen.

Full text of DOI:10.1016/j.poly.2019.01.057

Accepted Manuscript
Tetranuclear Schiff base copper (II) complexes: Syntheses, crystal structure,
DNA/protein binding and catecholase-like activity
Soumen Manna, Ennio Zangrando, Horst Puschmann, Subal Chandra Manna
PII:
S0277-5387(19)30079-8
https://doi.org/10.1016/j.poly.2019.01.057
POLY 13730
DOI:
Reference:
Polyhedron
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Accepted Date:
11 September 2018
8 January 2019
14 January 2019
Please cite this article as: S. Manna, E. Zangrando, H. Puschmann, S.C. Manna, Tetranuclear Schiff base copper
(II) complexes: Syntheses, crystal structure, DNA/protein binding and catecholase-like activity, Polyhedron (2019),
doi: https://doi.org/10.1016/j.poly.2019.01.057
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Tetranuclear Schiff base copper (II) complexes: Syntheses, crystal structure,
DNA/protein binding and catecholase-like activity
Soumen Manna,a Ennio Zangrando,b Horst Puschmann,c Subal Chandra Manna*,a
a
Department of Chemistry and Chemical Technology, Vidyasagar University, Midnapore
721102, West Bengal, India, E-mail: scmanna@mail.vidyasagar.ac.in, Fax: (91) (03222)
275329.
b Department of Pharmaceutical and Chemical Sciences, University of Trieste, 34127 Trieste,
Italy.
c Department of Chemistry, University of Durham, South Road, Durham DH1 3LE, UK.
Abstract
Two tetranuclear Schiff base copper(II) complexes, namely[Cu4(L)2(LH)2(H2O)2](ClO4)2·3H2O
(1) and [Cu4(L)2(LH)2(H2O)2](ClO4)·(tp)0.5·3H2O (2) (where H2L = 2-[(2-hydroxy-ethylimino)-
methyl]-6-methoxy-phenol, tp = terephthalate), were synthesized and characterized by X-ray
single crystal diffraction. Both complexes 1 and 2 are comprised of structurally similar
tetranuclear cationic [Cu4(L)2(LH)2(H2O)2]2+ species with a double open cubane core, in which
the two metal centers possess a square pyramidal environment and the other two exhibit a
slightly distorted octahedral coordination geometry. The interaction of complexes 1 and 2 with
calf thymus DNA (CT-DNA) was investigated using electronic absorption and fluorescence
studies, and the results showed that the complexes interact with CT-DNA with the related
intrinsic binding constants (Kib) of 1.8×106 and 1.1×107 L mol-1 for 1 and 2, respectively. Their
interactions with bovine serum albumin (BSA) and human serum albumin (HSA) were also
investigated and spectroscopic techniques showed that both complexes interact with these
proteins through a ground state association process. Using 3,5-di-tertbutylcatechol(3,5-DTBC) as

a model substrate, both complexes show catecholase-like activity, being able to oxidase 3,5-
DTBC to 3,5-di-tert-butylquinone (3,5-DTBQ) in the presence of oxygen.
Keywords: Tetranuclear Copper(II); Crystal structure; Catecholase activity; CT-DNA binding;
Serum albumin binding
1. Introduction
Study of transition metal coordination compounds of Schiff base ligands has attracted
interest in the last few decades, not only for their intriguing structures, but also for properties
manifested in the fields of magnetism [1], catalysis [2], fluorescence [3], medicinal chemistry
[4], etc. Among the 3d metal coordination compounds, polynuclear copper(II) complexes have
attracted consideration for their potential biological applications due to their antimicrobial [5],
antifungal [6], antidiabetic [7] and antitumor activities[8,5a], the latter showing a relatively low
toxicity in comparison to well-known platinum compounds [9]. Depending upon the Schiff base
ligand used, the copper(II) compounds, which have been widely reported in the literature, can
adopt various coordination numbers and geometries, such as square planar, square pyramidal,
trigonal bipyramidal and distorted octahedral. Tetranuclear copper complexes with a large
variety of structural motifs, such as cubane [10], pin-wheel [11], cyclic [12] and roof-shaped
[13], have been exploited with the aim to access correlations between their molecular structures
and magnetic behavior. In the last decade several synthetic copper(II) compounds have been
studied for developing new bioinspired catalysts [14]. In addition, multinuclear metal complexes
have been anticipated as synthetic metallonucleases to catalyze the hydrolysis of phosphodiester
bonds in DNA in order to develop novel metallopharmaceuticals [15]. For these purposes,

studies of the interaction of copper complexes with CT-DNA and serum albumins using
spectroscopic techniques represent a key point to develop possible copper metal based drugs.
Catechol oxidase, a copper enzyme with a type-3 active site, catalyzes the oxidation of a
wide range of o-diphenols (catechols) to their quinones. The catalytic activity of copper
complexes mimicking the catechol oxidase enzyme depends on many factors, such as
coordination environment, metal-metal distance, type of bridging ligand etc. [14]. A literature
study reveals that several mononuclear and dinuclear copper(II) complexes have been reported
showing catecholase-like activity [16], but there are only a limited number of copper(II)
complexes of high nuclearity [17].
In the present contribution, using the multidentate Schiff base ligand 2-[(2-hydroxy-
ethylimino)-methyl]-6-methoxy-phenol) (H2L), we have synthesized two tetranuclear copper(II)
complexes,
[Cu4(L)2(LH)2(H2O)2](ClO4)2·3H2O
(1)
and
[Cu4(L)2(LH)2(H2O)2](ClO4)·(tp)0.5·3H2O (2) (tp = terephthalate), both comprising a double open
cubane core structure. The interactions of complexes 1 and 2 with calf thymus DNA (CT-DNA),
bovine (BSA) and human (HSA) serum albumin were studied using spectroscopic techniques.
The catecholase activity of these complexes has also been investigated.
2. Experimental
2.1. Materials and physical measurements
Highly pure ethanolamine (98%) was purchased from Merck. Bovine serum albumin
(BSA), human serum albumin (HSA), ethidium bromide (EB) and calf thymus DNA (CT-DNA)
were purchased from Sigma Aldrich. All other reagent grade chemicals were commercially

available and used as received. The solvents used for spectroscopic studies were purified and
dried by standard procedures before use [18].
Elemental analyses (carbon, hydrogen and nitrogen) were performed using a Perkin–Elmer 240C
elemental analyzer. Electronic absorption spectra at room temperature were obtained with a
Shimadzu UV-1601 UV–vis spectrophotometer. Quartz cuvettes of 1 cm path length and 3 cm3
volume were used for all measurements. IR spectra were recorded as KBr pellets on a Bruker
Vector 22 FTIR spectrophotometer operating from 400 to 4000 cm-1. Emission spectra were
recorded on a Hitachi F-7000 spectrofluorimeter. The quantum yield was calculated according to
the reported method [19]. The ESI-MS spectra of the compounds were recorded in methanol
with an Agilent Q-TOF 6500 mass spectrometer. A BAS Epsilon electrochemical analyzer was
used for the cyclic voltammetric measurements.
2.2. Synthesis
Caution! Since perchlorate salts are potentially explosive, only small amounts of the materials
should be handled with care.
Complexes 1 and 2 have been synthesized adopting the procedures indicated in Scheme 1.

Scheme 1. Synthesis of complexes 1 and 2.
2.2.1. Synthesis of 2-[(2-hydroxy-ethylimino)-methyl]-6-methoxy-phenol (H2L)
The tridentate Schiff base 2-[(2-hydroxy-ethylimino)-methyl]-6-methoxy-phenol (H2L) was
prepared by the condensation reaction between 3-methoxysalicyldehyde (1 mmol, 0.152 g) and
2-aminoethanol (1 mmol, 0.06 g) in methanol (10 mL) according to the literature method [20].
2.2.2. Synthesis of [Cu4(L)2(LH)2(H2O)2](ClO4)2∙3(H2O) (1)
A methanolic solution (10 mL) of Cu(ClO4)2∙6H2O (0.5 mmol, 0.185 g) was added to a mixture
of H2L (0.5 mmol, 0.0976 g) and Et3N (0.5 mmol, 0.05 g) in methanol under stirring conditions.
Stirring was continued for 2 hrs and then the reaction mixture was filtered. The green colored
filtrate was left at room temperature and deep green crystals suitable for X-ray diffraction were
obtained after a few days.

Yield: 65%. Anal. Calcd for C40H56Cl2Cu4N4O25 (1317.95 g mol–1): C, 36.45; H, 4.28; N, 4.25
%. Found: C, 36.43; H, 4.26; N, 4.28 %. Selected FTIR bands (KBr, cm-1): 3419 (br), 2979 (m),
2940 (w), 2157 (br), 1639 (s), 1558 (vs), 1467 (m), 1416 (s), 1374 (w), 1302 (s), 1244 (m), 1218
(m), 1166 (w), 1079 (s), 1053 (w), 972 (m), 885 (w), 856 (w), 817 (w), 784 (w), 742 (m), 645
(w). UV-vis spectra [λmax, nm (ε, L mol-1 cm-1)] (CH3OH solution): 372 (1.029 × 106), 276 (4.117
× 106), 235 (8.114 × 106).
2.2.3. [Cu4(L)2(LH)2(H2O)2](ClO4)(tp)0.5∙(H2O)3 (2)
Complex 2 was synthesized following the procedure adopted for 1, but using an
additional methanolic solution (5 mL) of terephthalic acid (0.5 mmol, 0.83 g) and triethylamine
(1 mmol, 0.101 g). The stirring was continued for 2 hrs, then the deep green color solution was
filtered and the filtrate was kept at room temperature. After a few days deep green crystals
suitable for X-ray diffraction were collected by filtration. Yield: 70%. Anal. Calcd for
C44H57ClCu4N4O22.50 (1291.55 g mol–1): C, 40.91; H, 4.44; N, 4.33 %. Found: C, 40.89; H, 4.42;
N, 4.35 %. FTIR bands (KBr, cm-1): 3409 (br), 2972 (vs), 2940 (s), 2875 (w), 1639 (w), 1561
(vs), 1467 (s), 1429 (s), 1370 (s), 1296 (s), 1247 (m), 1215 (m), 1076 (s), 1005 (m), 969 (m), 920
(w), 878 (s), 814 (s), 739 (m), 649 (m), 509 (s). UV-Vis spectra [λmax, nm (ε, L mol-1 cm-1)]
(CH3OH solution): 373 (2.422 × 105), 271 (3.331 × 106), 233 (8.477 × 106), 212 (7.690 × 106).
2.4. Protein binding studies
Stock solutions of bovine serum albumin (BSA, 5.68 µM), human serum albumin (HSA, 1.85
µM) and the complexes (10 µM) were prepared in HEPES buffer solution (pH 7.2). The Stern-
Volmer constant (Ksv) and quenching rate constant (kq) were calculated using equations F0/F = 1

+ Ksv[complex] and Ksv = kqτ0, where F0 and F are the fluorescence intensities in the absence and
presence of the complex, and τ0 is the lifetime of serum albumin (10-8 s). To calculate the binding
constant of the compound with serum albumin, the Scatchard equation, log(Fo–F)/F = log Kb +
nlog[complex], has been used, where Kb is the binding constant of the compound with serum
albumin and n is the number of binding sites per albumin.
2.5. DNA binding experiments
2.5.1. Electronic absorption spectral study
The interaction between the copper complexes and CT-DNA was investigated using UV-vis
absorption spectral studies at room temperature. For both complexes the interaction has been
evaluated by the gradual addition of a 10 µL solution (6.66 µM) of CT-DNA to a fixed
concentration of the copper(II) complex (2 ml, 0.19 µM) in water. The intrinsic binding constant
(Kib) of the complexes with CT-DNA was determined using the equation:
ε ε ε ε ε ε
where [DNA] is the concentration of CT-DNA, is the extinction coefficient value of the
complex at a given CT-DNA concentration, ε and ε are the extinction coefficients of the free
complex in solution and when fully bound to CT-DNA, respectively. The plot of [DNA]/(ε
ε ) vs [DNA] gives a straight line with
and
as the slope and intercept,
ε ε
ε ε
respectively. The value of Kib was calculated from the ratio of the slope to the intercept.

2.5.2. Competitive EB-DNA binding fluorescence measurement
The competitive binding nature of ethidium bromide (EB) and the metal complex with CT-DNA
was investigated adopting a fluorometric method using an aqueous solution of EB bound CT-
DNA in HEPES buffer at room temperature. The fluorescence intensities at 614 nm (λex, 500
nm) of EB bound CT-DNA with increasing concentrations of the complex were recorded. The
quenching constants (Stern-Volmer constant, Ksv) were calculated using the Stern-Volmer
equation, F0/F = 1 + Ksv[complex], where F0 and F are the emission intensities in the absence and
presence of the copper(II) compound, Ksv is the Stern-Volmer constant and [complex] is the
copper(II) complex concentration.
2.6. Crystallographic data collection and refinement
Data collections for complexes 1 and 2 were carried out at 120 K with Mo-Kα radiation (λ =
0.71073 Å) on an Xcalibur diffractometer equipped with a Sapphire 3CCD camera. Cell
refinement, indexing and scaling of the data sets were done by using program CrysAlisPro [21].
The structures were solved by direct methods and subsequent Fourier analyses [22], and then
refined by the full-matrix least-squares method based on F2 with all observed reflections [22]. In
2, the perchlorate oxygen atoms and one water molecule were refined over two disordered
positions (occupancies of 0.598(9)/0.402(9) and 0.59(3)/0.41(3), respectively). An additional
residue was interpreted as a water molecule with half occupancy. All the calculations were
performed using the WinGX System, Ver 1.80.05 [23]. The molecular structures were obtained
with the graphical program Diamond [24]. Crystal data and details of the refinements are given
in Table 1.
3. Result and discussion

3.1. Crystal structure description
The single crystal X-ray structure determination shows that both complexes 1 and 2 are
comprised of structurally similar tetranuclear cationic species, [Cu4(L)2(HL)2(H2O)2]2+ (Fig.1),
which are counterbalanced by two perchlorate anions in 1, and perchlorate with terephthalate
anions in 2. Selected bond distances for the complexes are listed in Table 2. As the tetranuclear
cations are similar, the description is limited to complex 1. The cubane-like Cu4O4 core is
formed by four copper atoms and four oxygen donors from four Schiff base ligands, two of
which are deprotonated [HL]-, acting as µ2–η1:η1:η2:η1-O,N,O,O, the other di-deprotonated [L]2-,
acting as µ3–η1:η1:η3-O,N,O, and the overall geometry can be described as an open cage. In both
of these complexes the four Cu(II) atoms are not crystallographically equivalent, although the
species present a pseudo-two fold axis. The Cu1 and Cu3 copper centres exhibit a square
pyramidal geometry, being chelated by imino nitrogen, phenoxido and alcoholic oxygen atoms
(N1a/N1b, O2a/O2b, O1a/O1b for Cu1/Cu3, respectively) from two [HL]- ligands. The metals
complete the basal plane with the alkoxido oxygen atoms from two [L]2- ligands. The apical
position of the square pyramidal geometry at the Cu1 and Cu3 atoms is occupied by a water
molecule (O1w and O3w). On the other hand, the Cu2 and Cu4 atoms present an octahedral
coordination geometry formed by imino nitrogen, phenoxido and alkoxido oxygen atoms
(N1c/N1d, O2c/O2d, O1c/O1d for Cu2/Cu4, respectively) from the ligands [L]2-. The
coordination sphere is completed by phenoxo O2a and O2b donors from the ligands [HL]- and at
axial positions by another alkoxido atom, O1d/O1c, and the methoxy oxygen atom, O3a/O3b, for
Cu2/Cu4, respectively.
The basal coordination bond distances (Table 2) for the Cu1 and Cu3 atoms are between
1.910(5) and 2.048(5) Å, while the aqua ligand at the apical position is somewhat more distant,

at 2.235(5) and 2.262(5) Å, respectively. The Addison τ values [25] for the Cu1 and Cu3 centres
[0.235 and 0.121] indicate that the penta-coordination geometry is square pyramidal. The degree
of trigonality, τ, defined as (β–α)/60 (where β and α are the two largest coordination bond
angles), is zero for a perfect square pyramidal, while it becomes unity for ideal trigonal
bipyramidal geometry. The equatorial bond distances for the Cu2 and Cu4 centres are in the
range 1.900(5)-2.025(5) Å, and as expected, the axial bond lengths, between 2.416(5) and
2.554(5) Å, are longer than the equatorial bond values, due to Jahn–Teller distortion. In addition,
with the aim to outline the architecture of the cation, it is worth noting the rather long Cu1-O2b
and Cu3-O2a distances of 2.878 and 2.891 Å, respectively.
The data of Table 2 indicate comparable values of the distances (most within their e.s.d.’s) for
complexes 1 and 2, but the Cu1 and Cu3 atoms in the latter show minor deviations from an ideal
square pyramidal geometry, the τ parameters being 0.040 and 0.215, respectively.
The crystal packing shows lattice water molecules with perchlorate ions (compound 1, Fig.2) and
lattice water molecules with terephthalate ions (in 2, Fig.3) form layers through extended H-
bonding that, via additional hydrogen bond interactions with the complex cations, give rise to a
3D network in both compounds.
3.2. Electronic and emission spectra of complexes 1 and 2
The electronic spectra of complexes 1 and 2 were recorded in methanol (Fig. 1S, Table
1S). Compound 1 shows significant transitions at 235 nm (ε ~ 8.11 × 106 L mol-1 cm-1), 276 nm
(ε ~ 4.11 × 106 L mol-1 cm-1) and 372 nm (ε ~ 1.02 × 106 L mol-1 cm-1), and the spectral
transitions of compound 2 are at 212 nm (ε ~ 7.75 × 106 L mol-1 cm-1), 233 nm (ε ~ 8.47 × 106 L
mol-1 cm-1), 271 nm (ε ~ 3.33 × 106 L mol-1 cm-1) and 373 nm (ε ~ 2.42 × 105 L mol-1 cm-1).

On excitation at 276 nm, compound 1 exhibits emission bands at 357 and 313 nm in
methanol at room temperature and for compound 2 excitation at 271 nm results in emission
bands at 361 and 313 nm (Fig. 2S, Table 1S).
3.3. Infrared spectra of complexes 1 and 2
The IR spectra show broad bands at around 3425 cm-1 for complex 1 and 3415 cm-1 for complex
2, assigned to the ν(OH) stretching vibration of the lattice water molecules (Fig. 3S, Table 2S).
In addition, bands at 513 and 817 cm-1 for complex 1, and 509 and 814 cm-1 for 2 indicate the
presence of coordinated water molecules [26]. The bands at 1416 (for 1) and 1429 cm-1 (for 2)
are due to the azomethine (C=N) group. The sharp peaks at 1079 (for 1) and 1076 cm-1 (for 2)
give evidence for the presence of free perchlorate ions [26]. The bands at 2979 cm-1 for 1 and
2972 cm-1 for 2 are due to aromatic C-H stretching vibrations, and the bands at 2940 cm-1 for
both compounds correspond to aliphatic ν(C-H) vibrations. Bands at 1165 and 1215 cm-1 for 1
and 2, respectively, are assigned to ν(O–CH3) vibrations. For 2, the stretching frequencies of the
-
-
free carboxylate ion, νa(CO2 ) and νs(CO2 ), appear at 1561 and 1467 cm-1, respectively.
3.4. ESI-MS spectra of complexes 1 and 2
The ESI mass spectra (Figs. 4S and 5S) of complexes 1 and 2 were recorded in methanol. The
mass spectra show peaks at m/z = 1065.82 (calc. 1066.03) and 1065.90 (calc. 1066.03) for
complexes 1 and 2, respectively. These peaks correspond to the species [C40H48N4O14Cu4]+H+
[calc. m/z = 1064.02], clearly indicating that the tetranuclear composition of complexes is
maintained in methanolic solution.
3.5. Catechol oxidase studies of complexes 1 and 2

3,5-Di-tert-butylcatechol (3,5-DTBC) is generally used as substrate to evaluate the
catecholase activity of coordination compounds since the oxidation product, 3,5-di-tert-
butylquinone (3,5-DTBQ), is highly stable and gives a characteristic absorption band at 400 nm
(ε = 1900 M−1 cm−1) in methanol. To verify the catecholase-like activity of complexes 1 and 2, a
10-4 M solution of each complex was treated with a 100-fold concentrated solution of 3,5-DTBC
and the reaction was followed by recording the UV-vis spectra of the mixture under aerobic
conditions at intervals of 5 minutes up to 90 min. These spectral changes, after mixing 3,5-
DTBC with complexes 1 and 2, are shown in Figs. 4 and 6S, respectively.
The rate constant for the complex-substrate mixture was determined by the initial rate
method from the plot of the optical density versus time [Figs. 7S and 8S]. The change of the rate
of reaction with the concentration of substrate is shown in Fig. 5, which allows the evaluation
that at a low concentration of 3,5-DTBC the reaction rate is first order, whereas saturation
kinetics are observed at higher concentrations.
The rate versus concentration of the substrate (Fig. 5) was then analyzed on the basis of the
Michaelis-Menten approach of enzymatic kinetics to get a Lineweaver-Burk plot (Fig. 6), as well
as the parameter values of the maximum initial rate (Vmax), Michaelis-Menten constant (KM) and
turnover number (Kcat) (Table 3). Compound 1 showed a higher catecholase-like activity (kcat
=
113 h−1) with respect to compound 2 (kcat = 97 h−1), which may be tentatively be referred to the
larger distortions detected in the coordination geometry of the former complex [trigonality τ
values of 0.235 and 0.121 for complex 1 and 0.040 and 0.215 for 2], or to the templating effects
of the counter ions.
In order to elucidate the mechanism and possible complex-substrate intermediate, we have
recorded the ESI-MS spectra of 1:100 mixture of each complex with 3,5-DTBC (Figs. 9S and

10S). The peaks at m/z = 196.22 (for 1) and 196.21 (for 2) are due to protonation of the ligand
[H2L]+H+. The formation of a complex-substrate intermediate species was identified by peaks at
m/z = 477.50 (for 1) and 477.44 (for 2) [17]. The peak at m/z = 243 can be assigned to the
sodium-quinone aggregate [3,5-DTBQ]+Na+. The molecular oxygen oxidising 3,5-DTBC to 3,5-
DTBQ in this process is converted to H2O2, the formation of which is confirmed by the blue
color observed when starch and potassium iodide are added to the mixture of the complex and
3,5-DTBC. It is interesting to note that such coloration is not observed in the absence of the
complex. A probable mechanistic path for the formation of H2O2 as a by-product during the
oxidation of 3,5-DTBC to 3,5-DTBQ catalyzed by a copper(II) compound was suggested by
Chyn and Urbach [27]. Based on the ESI-MS spectra and considering the formation of H2O2 as a
by-product we have proposed a possible mechanism of the reaction, shown in Scheme 1S. The
catalytic reaction starts with the formation of intermediate I, then one copper centre acquires one
electron from 3,5-DTBC to form the intermediate II [m/z = 477.50 (for 1) and 477.44 (for 2)].
3.6. Protein binding studies
In a fluorometric study, 10 µL of an aqueous complex solution (10 µM) were gradually
added to 3 ml of serum albumin solutions (BSA, 5.68 µM; HSA, 1.85 µM) and the quenching of
the emission intensities at 340 nm (λex, 278 nm) for BSA and at 330 nm (λex, 278 nm) for HSA
was recorded. Upon gradually increasing the complex concentration, the fluorescence intensity
of BSA and HSA significantly decreased (Figs. 7-8 and 11S-12S). The Ksv values were
calculated using the Stern-Volmer plot (Fig. 13S for BSA, and Fig. 14S for HSA). The
calculated binding constants (kb) for the interaction of the complex with the serum albumins are

(4.21 × 107) 3.74 L mol-1 (1-BSA), (8.53 × 103 1.90 L mol-1(1-HSA), (2.46 × 106 2.31 L
mol-1 (2-BSA) and (8.25 × 103 2.76 L mol-1 (2-HSA) (Table 4).
The quenching of the fluorescence intensity of BSA and HAS solutions upon addition of
the complexes is indicative of the association of serum albumins to the metal species.
Fluorescence quenching may occur either by a static or dynamic mode. From the UV-vis spectral
study it was inferred that the absorption spectra of BSA/HSA are influenced by the addition of
the complexes and the phenomenon evidenced the existence of a static interaction between the
proteins and complexes studied.
3.7. DNA binding studies of the complexes
An aqueous solution of complex 1 showed bands at 229, 274 and 370 nm. Fig. 9 shows the
changes in the electronic absorption spectra of complex 1 upon gradual addition of 10 µL (6.66
µM) of an aqueous solution of CT-DNA to a 2 ml of complex aqueous solution (0.19 µM). The
absorbance band at 274 nm gradually increased with a blue shift up to 269 nm (Fig. 9). The
isobestic points at 229, 241 and 348 nm in Fig. 9 indicate the presence of more than two species
in solution [28]. Complex 2 exhibited bands at 227, 273 and 364 nm, and those at 273 and 364
nm gradually increased with a blue shift up to 270 and 350 nm, respectively (Fig. 15S), upon the
gradual addition of the CT-DNA solution. The calculated values of the intrinsic binding constant
(Kib) are 1.8 × 106 and 1.1 × 107 L mol-1 for 1 and 2, respectively.
Ethidium bromide (EtBr = 3,8-diamino-5-ethyl-6-phenyl phenanthridinium bromide)
shows fluorescence of an orange color when exposed to ultra violet radiation. EtBr bound to CT-
DNA shows an emission band at 614 nm on excitation at 500 nm. Addition of a compound
capable of interacting with CT-DNA results in quenching of the fluorescence intensity. In a

fluorometric interaction study, a reduction of the fluorescence intensity was detected (Figs. 10
and 16S) upon gradual addition of 10 µL of a 10 µM aqueous solution of the complex to the
aqueous solution of EtBr bound CT-DNA (2 ml 7.87 µM). This observation suggests that the
complexes displaced EtBr molecules from the DNA binding sites. From the Stern-Volmer
equation, linear relationships were obtained from the titration of EtBr-CT-DNA using the
complexes as a quencher. The calculated values of the binding constant (Ksv) are 7.78 × 105 and
8.61 × 105 L mol-1 for 1 and 2, respectively. The quenching of fluorescence occurs due to a
decrease in the number of CT-DNA binding sites available for EtBr. The fluorescence quenching
observed in the presence of the complexes may be due to intercalation of the complexes in the
CT-DNA helices.
3.8. Cyclic voltammetry studies
The electrochemical behavior of complexes 1 and 2 was investigated by cyclic voltammetry in
methanol solution in the electric potential range from 0.0 to 0.6 V, using tetraethyl ammonium
perchlorate (TEAM) as the supporting electrolyte (0.1 M). The cyclic voltammograms showed
(Fig. 17S) an irreversible oxidation at 0.43 and 0.41 V for 1 and 2, respectively, which was
assigned to the CuII/CuIII process [29].
4. Conclusion
In summary, we have presented here the synthesis, crystal structure, catecholase-like activity and
study of the interactions with BSA/HSA and CT-DNA of two copper(II) compounds synthesized
with 2-[(2-hydroxy-ethylimino)-methyl]-6-methoxy-phenol as a chelating ligand. Both

compounds are tetranuclear with a double open cubane core structure. Electronic absorption and
fluorescence spectroscopic studies indicate that both complexes interact with CT-DNA and
serum albumins. In addition, complexes 1 and 2 are active towards the catalytic oxidation of 3,5-
DTBC to 3,5-DTBQ in the presence of molecular oxygen, and 1 discloses a higher catalytic
activity, likely induced by slightly different distortions of the copper center in comparison to 2,
or to templating effects of the counter ions.
Acknowledgements
The authors gratefully acknowledge the financial assistance given by the CSIR, Government of
India to Dr. Subal Chandra Manna (Project No. 01 (2743)/13/EMR-II). Dr. S. C. Manna thanks
UGC-SAP and DST-FIST New Delhi and Vidyasagar University for infrastructural facilities.
Supplementary information
CCDC1852481 and 1852480 contain the supplementary crystallographic data for complexes 1
and 2, respectively. These data can be obtained free of charge via
http://www.ccdc.cam.ac.uk/conts/retrieving.html, or from the Cambridge Crystallographic Data
Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: (+44) 1223-336-033; or e-mail:
deposit@ccdc.cam.ac.uk. IR, electronic absorption, emission spectral results, figures of crystal
structures, protein binding and CT-DNA binding are provided as supporting information.
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Table 1. Crystal data and details of the structure refinement of complexes 1 and 2.
Complex
1
2
Empirical formula
C40H56Cl2Cu4N4O25 C44H57ClCu4N4O22.50
Formula mass, g mol–1 1317.95
1291.55
Monoclinic
P 21/n
14.6377(3)
21.7677(5)
16.8163(4)
108.225(2)
5089.4(2)
4
Crystal system
Space group
a, Å
Monoclinic
P 21/c
11.8650(6)
23.6691(7)
18.4922(8)
98.048(4)
5142.1(4)
4
b, Å
c, Å
β, deg
V, Å3
Z
D(calcd), g cm–3
1.702
1.686
(Mo-K), mm–1
F(000)
1.825
2696
1.788
2648
Theta range, deg
No. of collected data
No. of unique data
Rint
3.11 - 26.02
44411
2.55 - 28.28
62449
10110
0.0849
7502
12525
0.0599
Observed reflns [I>
2σ(I)]
9269
Goodness of fit (F2)
1.188
692
1.037
Parameters refined
729
0.0583, 0.1232
0.908, –0.752
R1, wR2 (I >2σ(I)) [a] 0.0772, 0.1549
Residuals, e Å–3
0.837, –0.767
½
[a]R1(Fo) = Fo–Fc / Fo, wR2(Fo2) = [w (Fo2 – Fc 2) 2/ w (Fo2) 2 ]

Table 2. Selected bond lengths (Å) and intermetallic distances for complexes 1 and 2.
1
2
Cu(1)-N(1a)
Cu(1)-O(1a)
Cu(1)-O(2a)
Cu(1)-O(1d)
Cu(1)-O(1w)
Cu(2)-N(1c)
Cu(2)-O(1c)
Cu(2)-O(2c)
Cu(2)-O(2a)
Cu(2)-O(3a)
Cu(2)-O(1d)
Cu(3)-N(1b)
Cu(3)-O(1b)
Cu(3)-O(1c)
Cu(3)-O(2b)
Cu(3)-O(3w)
Cu(4)-N(1d)
Cu(4)-O(1d)
Cu(4)-O(2d)
Cu(4)-O(2b)
Cu(4)-O(3b)
Cu(4)-O(1c)
1.926(7)
2.048(5)
1.949(5)
1.923(5)
2.235(5)
1.922(7)
1.962(5)
1.919(5)
2.025(5)
2.432(5)
2.488(5)
1.937(6)
2.007(5)
1.910(5)
1.951(5)
2.262(5)
1.930(6)
1.987(5)
1.900(5)
1.996(5)
2.416(5)
2.554(5)
1.946(4)
2.003(3)
1.957(3)
1.923(3)
2.290(3)
1.932(4)
1.954(3)
1.901(3)
1.995(3)
2.428(3)
2.532(3)
1.941(4)
1.994(3)
1.932(3)
1.946(3)
2.287(3)
1.934(4)
1.957(3)
1.896(3)
1.996(3)
2.432(3)
2.434(3)
Cu(1)-Cu(2)
Cu(1)-Cu(3)
Cu(1)-Cu(4)
Cu(2)-Cu(3)
Cu(2)-Cu(4)
Cu(3)-Cu(4)
3.2030(13)
3.9033(13)
3.2885(12)
3.3046(12)
3.3565(12)
3.1605(12)
3.1814(7)
3.8058(7)
3.3507(7)
3.2862(7)
3.2344(7)
3.1587(7)
Table 3. Kinetics parameters for the catecholase activities of complexes 1 and 2 in methanol.
Complex
Vmax (M min-1)
6.289×10-7
KM (M)
Kcat (h-1)
113
2.045×10-3
1.202×10-3
1
2
5.406×10-7
97

Table 4. Kinetics parameters for the protein binding activities of the complexes 1 and 2.
Complex
1-BSA
2-BSA
1-HSA
2-HSA
kb (L mol-1)
n
ksv(L mol-1)
(5.54 ×106
(1.54 ×106
(1.64 ×106
(1.26 ×106
Kq(L mol-1sec-1)
(5.54 )×1014
(1.54 )×1014
(1.64 )×1014
(1.26 )×1014
(4.21×107)
(2.46×106 2.31
(8.53×103 1.90
(8.25×103 2.76
(1.13
(1.02
(0.67
(0.66
Captions of the figures
Fig.1.Molecular structure of complex 2 with labels for the hetero-atoms. The same scheme also
applies to complex 1.
Fig.2. Layered structure in the crystal packing of complex 1 formed by ClO4 anions and water
molecules connected by H-bonds.
Fig.3. Layered structure in crystal packing of compound 2 formed by terephthalate anions and
water molecules connected by H-bonds.
Fig.4. Increase of the absorbance around 400 nm after the addition of 100 equiv. of 3,5-DTBC to
a methanolic solution of complex 1. The spectra were recorded at intervals of 5 min.
Fig.5. Initial rates vs substrate concentration for the 3,5-DTBC to 3,5-DTBQ oxidation reaction
catalyzed by complexes 1 and 2 in methanol.
Fig.6. Lineweaver-Burk plot of complexes 1 and 2.
Fig.7. Change of the emission spectra of BSA (3 ml, 5.68 µM aqueous solution) upon gradual
addition of 10 µL of an aqueous solution (10 µM) of complex 1 at room temperature. The arrow
indicates the increase of the complex concentration.

Fig.8. Change of the emission spectra of HSA (3 ml, 1.53 µM aqueous solution) upon gradual
addition of 10 µL of an aqueous solution (10 µM) of complex 1 at room temperature. The arrow
indicates the increase of the complex concentration.
Fig.9. Change of the electronic absorption spectra of complex 1 (2 ml, 0.19 µM) upon the
gradual addition of 10 µL of an aqueous solution (6.66 µM) of CT-DNA. Inset: Plot of [DNA] /
(εa-εf) vs [DNA]. The arrow shows the changes in absorbance with the increase of DNA
concentration.
Fig. 10. Fluorescence quenching curves of EB bound to CT-DNA upon gradual addition of
complex 1 (10 µL, 10 µM). Inset: Stern-Volmer plot of the fluorescence titration.
Fig.1

Fig.2
Fig.3

Fig.4
Fig.5

Fig.6
Fig.7

Fig.8
Fig.9

Fig. 10

Two tetranuclear Schiff base coordinated copper(II) complexes have been synthesized and
structurally characterized. The catecholase-like activity and the interaction of the complexes with
DNA/proteins were studied using spectroscopic techniques.